1. Field of the Invention
The present invention is in the general field of the catabolic pathway of chitin and generally relates to genes encoding enzymes for cleaving chitin into its component parts.
2. Description of the Related Art
At least 18 species of Vibrionaceae are chitinolytic. Six are human pathogens, including V. furnissii, V. cholerae and V. parahaemolyticus. A brief review (1) entitled "Cholera, Copepods, and Chitinase" describes the relationships between the Vibrios, zooplankton, annual cycles of the bacteria, the invertebrates, and human disease such as food poisoning and endemic cholera. One important element in the epidemiology (2) is that V. cholerae adhering to chitin particles are protected from acid (equivalent to the stomach acid barrier) which kills almost all of the free-living organisms. This protection is explained by the fact that virtually all of the microbes in zooplankton "burrow" into the organism, and are not exposed to the medium (3).
Chitin and chitosan are commercial products used (especially in Japan) in medicine, agriculture, and for waste and water treatment. The polymers are used as wound dressing synthetic skin, drug delivery systems, sutures, to make contact lenses, as anticholesteremic agents, bactericidal agents, etc. (4). Chitin sutures are slowly degraded by lysozyme, and eventually absorbed, although nothing is known of the fate of the products, (GlcNAc).sub.n. (GlcNAc).sub.6 is claimed to be a potent anti-metastatic agent against mouse bearing Lewis lung carcinoma, and (GlcNAc).sub.n activate macrophages and the immune system.
Although chitinase activities were recognized early in this century (5), the first reports on the stepwise enzymatic degradation of the polymer appear to be those of Zechmeister and Toth (6) who chromatographed extracts of almond emulsin, and of the snail, Helix pomatia, and separated an exo and an endoenzyme from each. The chitinase or "polysaccharidase" converted particulate chitin to the disaccharide, N,N'-diacetylchitobiose, (GlcNAc).sub.2, and the "chitobiase," or .beta.-N-acetylglucosaminidase (.beta.-GlcNAcidase), hydrolyzed the disaccharide to GlcNAc. Chitin degradation continues to be intensively studied (4,5,7). Chitinases and chitobiases are found in bacteria, fungi, plants, and animals (vertebrates and invertebrates). The structural genes encoding a number of these enzymes and some of their regulatory regions have been cloned and sequenced (5,8-21). These data show that some organisms are capable of expressing multiple chitinases, but the pathway of chitin degradation is essentially the same as that proposed in the original studies (6), i.e., virtually all investigators agree that only two enzymes are required to degrade chitin to GlcNAc (5,7). The results of the present invention with Vibrio furnissii differ markedly from this concept. This organism not only expresses unique hexosaminidases, but we estimate that more than two dozen proteins are required for utilization of the polysaccharide (conversion to GlCNAc-6-P).
Despite early interest in chitin utilization by marine bacteria, there are few reports on the pathway in these organisms. A chitinase gene was cloned from Aeromonas hydrophila (an aquatic bacterium) into E. coli (22); the enzyme is normally secreted by the Aeromonas into the medium, but in the transformant it traversed only the inner membrane. Zyskind et al. (23,24) cloned the .beta.-GlcNAcidase gene from V. harveyi into E. coli, found that it was transported to the outer membrane after cleavage of a signal sequence, and that the gene sequence was similar to that of the .alpha.-chain of human .beta.-hexosaminidase (5). In V. harveyi, the .beta.-GlcNAcidase is induced by (GlcNAc).sub.2. A .beta.-GlcNAcidase gene has also been cloned from V. vulnificus (25), and these researchers suggest that this single enzyme is responsible for the complete degradation of chitin to GlcNAc, although the E. coli transformant is unable to clear chitin on chitin/agar plates. The chitobiase gene from V. parahaemolyticus was cloned into E. coli and the enzyme purified to homogeneity (26). The purified preparation showed four closely stacked bands, which the authors speculate may result from post-translational processing at the C-terminus; the hexosaminidase was active over the pH range 4-10. Laine also reports in an Abstract from a recent meeting (27) that his laboratory has cloned a chitinase gene from V. parahaemolyticus; the chitinase is secreted by the E. coli transformant.
While chitin and chitosan have been used commercially for various purposes for many years (4), the respective oligosaccharides have only recently been shown to be physiologically active. Chitin oligosaccharides (derivatized at the non-reducing end with a fatty acyl group) are signals generated by the soil bacterial genus Rhizobium, and recognized by host leguminous plants so that nitrogen fixing nodules are formed (51). Chitosan and chitin oligosaccharides induce pisatin and as many as 20 disease resistance response proteins in pea tissue and inhibit the growth of some fungal pathogens. GlcNAc and (GlcNAc).sub.2 were inactive, the trimer was slightly active, and the tetramer and pentamer were moderately active, both as antifungicides and pisatin elicitors (52,53). (GlcNAc).sub.6 is a potent antimetastatic agent against mouse bearing Lewis lung carcinoma, and (GlcNAc).sub.n activate macrophages and the immune system (13). The disaccharide, (GlcNAc).sub.2 is linked to the amide group of asparagine in a large number of glycoproteins, such as those found in the blood. The disaccharide is the core to which the oligosaccharide chains of these glycoproteins are attached. Enzymes that hydrolyze the glycoprotein or glycopeptides by splitting the disaccharide (e.g., Endo A and H) or the asparagine amide (releasing the oligosaccharide) are of considerable commercial significance since they are useful for analysis and structure determination of these important macromolecules.
It is important to emphasize that the plant defense mechanisms are induced by the elicitor oligosaccharides. The multitude of proteins in the V. furnissii chitin catabolic cascade are likewise induced, and induction is differential. That is, higher (GlcNAc).sub.n oligomers induce the extracellular chitinases, (GlcNAc).sub.2 induces a large number of proteins required for its catabolism but not the chitinases, and GlcNAc induces those proteins required for its metabolism but not the others. More importantly for present purposes, GlcNAc represses expression of the enzymes induced by (GlcNAc).sub.2 even when the latter is present in the medium, and (GlcNAc).sub.2 appears to repress expression of the chitinases. The biological activities of chitin and chitosan oligosaccharides may be expressed by individual oligomers, but not by mixtures of oligomers, especially by mixtures containing the lower molecular weight oligosaccharides.
The oligosaccharides have use in agriculture (e.g., to induce disease resistance) and in medicine. The costs of the commercially available oligosaccharides are prohibitive. While practical grade chitin costs from $22-49 per kilogram, the pure oligosaccharides cost from $5/mg (for (GlcNAc).sub.2) to about $15/mg (for (GlcNAc).sub.6). The problem can be illustrated with one example. (GlcNAc).sub.2 induces a large number of important proteins and enzymes in V. furnissii, whereas (GlcNAc).sub.5 and (GlcNAc).sub.6 induce others (48). The minimum concentration of (GlcNAc).sub.2 required for maximum induction is 0.6 mM in the growth medium (containing lactate or glycerol to spare the disaccharide). Thus, 0.6 mM (GlcNAc).sub.2 for one liter of medium would cost $1,270 and yield about 250 mg of induced cells (dry weight) and a few .mu.g of each enzyme. For the experiments involving (GlcNAc).sub.6 at 0.6 mM, the cost would be $11,000 per liter!
The procedure for making these oligomers explains their cost. The first method for isolating chitosan oligomers was developed in the laboratory of the present inventors (54), as well as the method for their quantitative N-acetylation (55,56). The same methods are still being used commercially as indicated in the Seikagaku America, Inc., catalogue. Briefly, the procedure is as follows: purified chitin is completely deacetylated by fusion with KOH pellets under N.sub.2, giving chitosan. The latter is purified by "recrystallization" 12 times to remove colored impurities, and partially hydrolyzed in 10.5N HCl at 53.degree. C. for 72 h. The hydrolysate is applied to an ion-exchange column and eluted with a 0 to 4.2M HCl gradient. In this procedure, 5 g of chitosan were used, the ion exchange column contained 1 liter of resin, and 500 ml fractions were collected (total volume, 60 liters!). While the resolution from monomer to at least the pentamer was very good, it is obvious that the method is very limited with respect to quantity. For example, 244 mg of (GlcNH.sub.2).sub.5 were obtained. Following quantitative N-acetylation with acetic anhydride, this quantity of material is sufficient for one 400 ml V. furnissii induction/growth experiment of the type described above.
The major problem in isolating large quantities of pure oligosaccharides are the limitations in resolving mixtures of these compounds. Even E-chitinase, which hydrolyzes chitin primarily to (GlcNAc).sub.2, yields significant quantities of GlcNAc. Wild type and genetically engineered V. furnissii and E. coli cells are used to remove contaminants. The lower six carbon atoms of sialic acid have the configuration of N-acetylmannosamine (not previously recognized as a natural sugar), not GlcNAc as reported (57-59). To study the metabolism, especially the enzymatic synthesis of sialic acid, requires substrate quantities of N-acetylmannosamine (ManNAc). The chemical synthesis of ManNAc is tedious and gives small amounts of material. The problem was solved (60) by alkaline epimerization of 25 to 100 g quantities of N-acetylglucosamine; the equilibrium mixture contained 80% GlcNAc and 20% ManNAc. Part of the GlcNAc crystallized when the solution was concentrated, and the remainder (5 to 20 g, depending on the scale) was removed with E. coli cells induced to catabolize GlcNAc. To illustrate the power of the method, 200 mg of E. coli cells (dry weight) obtained from 1 liter of culture were sufficient to completely remove all of the GlcNAc from the 25 g GlcNAc epimerization mixture in 4 h at 37.degree. C. After the incubation, the mixture was deproteinized with Ba(OH).sub.2 and ZnSO.sub.4, deionized, and pure ManNAc crystallized from the concentrated supernatant fluid in 70% yield (3.5 g of the 5 g formed in the epimerization reaction). Yields up to 80% were obtained from the 100 g reaction. In studies on the physical properties of the periplasmic space in E. coli and Salmonella typhimurium (61), it was necessary to remove traces of glucose and fructose from commercial (labeled and unlabeled) sucrose. The same methodology was successfully employed.
The preparation of the chitin oligosaccharides is based on similar procedures, i.e., a combination of partial hydrolysis of chitin to yield a mixture of soluble oligomers, followed by treatment with appropriate enzymes and/or mutant or transformed cells to resolve the mixtures and to obtain single products, or of desired mixtures, such as (GlcNAc).sub.4 and (GlcNAc).sub.5.